Orotidine 5'-monophosphate decarboxylase (ODCase) is a key enzyme in the de novo pyrimidine biosynthetic pathway. This pathway is essential for the biosynthesis of building blocks for both DNA and RNA, and thus inhibitors of ODCase could have significant therapeutic value. Despite recent significant advances, it is not understood what factors are important in binding to and catalysis by ODCase. The lack of such knowledge is a critical defecit because understanding these factors is essential to the design of potent inhibitors for this enzyme. Our long-range goal is to understand the catalytic mechanism of ODCase and to design potent inhibitors of ODCase based on the mechanism. The objective of this application, which is a step in pursuit of that goal, is the identification of structural components of the substrate and its analogs that are important for binding and catalysis. The central hypothesis of the application is that the zwitterionic content (determined by proton affinity) is the most important factor in determining the rate of decarboxylation in model and enzyme-catalyzed reactions as well as the tightness of binding and that binding energy from remote binding sites is utilized by ODCase to catalyze the reaction. The hypothesis will be tested by pursuing the following two specific aims: 1) Identify the role played by the zwitterionic intermediate in the model and enzymatic decarboxylation of substrate analogs and identify the structural components on the pyrimidine ring of the substrate and analogs that are important for binding and catalysis;and 2) Determine the role of remote binding sites (the hydroxy groups on the ribose moiety) toward binding and catalysis. The proposed research is innovative because it combines model and enzymatic studies to identify the role of zwitterioic structures in the reaction mechanism and to determine the factors important for binding and catalysis. This contribution is significant, therefore, because it will provide an understanding of how enzymes utilize remote binding energy in catalysis in general, and the mechanism as well as the design of potent and selective inhibitors of ODCase, in particular. Relevance to Public Health: It is of great interest to develop potent inhibitors of ODCase because it is part of the nucleic acid biosynthesis machinery. Inhibition of the biosynthesis of these building blocks has been exploited in cancer chemotherapy. Many other pyrimidine antagonists have been shown to be useful in antitumor and antiviral chemotherapy